CN114196638B - Adeno-associated virus purification kit and purification method - Google Patents

Adeno-associated virus purification kit and purification method Download PDF

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CN114196638B
CN114196638B CN202111621847.8A CN202111621847A CN114196638B CN 114196638 B CN114196638 B CN 114196638B CN 202111621847 A CN202111621847 A CN 202111621847A CN 114196638 B CN114196638 B CN 114196638B
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antibody
adeno
associated virus
aav
microbeads
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CN114196638A (en
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谢洪学
王美美
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Wuhan Meibojin Biotechnology Co ltd
West Anhui University
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West Anhui University
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14151—Methods of production or purification of viral material

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Abstract

The invention relates to an adeno-associated virus purification kit, which comprises a lysis-balance buffer solution, a first antibody, anchoring microbeads and an eluent; the first antibody is a nano antibody and is provided with a heavy chain variable region and an Fc region, and the sequence of the heavy chain variable region is shown as SEQ ID NO. 1; the antibody-conjugated microbeads are microbeads conjugated with a second antibody, which is an antibody against the Fc region. The present invention screens and prepares antibodies with high specificity and affinity for AAV, and the antibodies have a specific pH activity range, making them easy to use for isolation of AAV. The AAV purification kit prepared by utilizing the characteristics of the AAV antibody can rapidly purify adeno-associated virus by a centrifugal separation or magnetic separation mode, and improves the infection titer of AAV obtained by separation.

Description

Adeno-associated virus purification kit and purification method
Technical Field
The invention relates to the field of biological medicine, in particular to an adeno-associated virus purification kit and a purification method.
Background
In biological and medical research and industrialization, transgenic technology is an important research content and means of use, and has been in rapid development, and vector virus technology developed rapidly in recent years is a widely used transgenic technology. The use of various vector viruses has resulted in a large market, including cloning, packaging, preparation, purification, etc. of the viruses, which are commercially available products and services. Adeno-associated virus (AAV) is a commonly used vector virus, and has been widely used in vaccine, gene therapy, scientific research, and the like. The demand for adeno-associated viruses is also increasing at present due to the demands of production and research.
The current adeno-associated virus preparation and purification technology is relatively lagging, and the purification process is relatively complex because the virus remains in the packaging cells after packaging. The method for purifying adeno-associated virus in laboratory mainly adopts an ultracentrifugation technology, and the method firstly needs an ultracentrifuge, and the operation process is complex, time-consuming and labor-consuming. We used the Cell Biolabs (USA) and Biominga (USA) adeno-associated virus purification kits in tandem with the laboratory of university of California, san Francisco, jennifer Whistler, but failed because of too low a recovery. Similar products to Cell Biolabs and biomega use the resin adsorption method. In addition, viralur corporation (united states) provides an adeno-associated virus purification kit based on a filtration membrane adsorption method, which has not been widely used because the filtration membrane adsorption method has low specificity for adsorption of adeno-associated virus, has limited adsorption capacity, and has not particularly good purification effect. Specific antibodies have been developed by some researchers to be carried on solid substrates to increase adsorption specificity for AAV. However, the use of specific antibodies presents new problems, such as difficulty in washing off AAV particles during elution, low recovery, and the possibility of elution of antibodies with AAV particles during elution, affecting AAV particle purity.
Thus, there is a need for new methods and purification reagent products for adeno-associated virus purification.
Disclosure of Invention
In order to solve the problems, the invention provides an adeno-associated virus purification kit, which comprises a lysis-balancing buffer solution, a first antibody, anchoring microbeads and an eluent;
the first antibody is a nano antibody and is provided with a heavy chain variable region and an Fc region, and the sequence of the heavy chain variable region is shown as SEQ ID NO. 1;
the antibody-conjugated microbeads are microbeads conjugated with a second antibody, which is an antibody against the Fc region.
The present invention enables rapid anchoring and elution of AAV by developing a first antibody to AAV, and a second antibody to the first antibody, and coupling the second antibody to microbeads. The kit of the invention carries out AAV purification by a centrifugal or magnetic method, thereby greatly accelerating the purification speed in production and scientific experiments, and simultaneously, the kit of the invention can provide AAV solution with higher infection titer.
In a specific embodiment, the lysis-equilibration buffer is a tris buffer at pH 8-8.5 containing tween 20 at a concentration of 0.05% to 0.1% and NP40 at a concentration of 0.2% to 0.3%. The specific affinity of the primary antibody to AAV is high at pH 8-8.5, which facilitates anchoring of AAV to a solid substrate such as microbeads.
In a specific embodiment, the eluent is a citric acid buffer of pH 4-5 containing 500-100 mM sodium chloride. The pH of 4-5 and sodium chloride concentration of 50-100 mM facilitate elution of AAV from the primary antibody while maintaining the primary antibody still anchored to the microbeads.
In a specific embodiment, the Fc region is a mouse Fc region.
In a specific embodiment, the second antibody is a nanobody, and the heavy chain variable region sequence is shown in SEQ ID NO. 5.
In a specific embodiment, the antibody-coupled microbeads are agarose microbeads or magnetic beads.
The invention also discloses a method for purifying the adeno-associated virus, which comprises the step of purifying the adeno-associated virus by using the adeno-associated virus purification kit.
In a specific embodiment, the method comprises the steps of:
s1: collecting cells containing adeno-associated virus, disrupting the cells using the lysis equilibration buffer, and centrifuging to obtain a supernatant containing adeno-associated virus;
s2: adding the first antibody into the supernatant, and uniformly mixing to obtain a first antibody-supernatant;
s3: adding the antibody-coupled microbeads into the first antibody-supernatant, and uniformly mixing;
s4: separating the antibody-conjugated microbeads from the first antibody-supernatant;
s5: eluting the antibody-coupled microbeads with the eluent to obtain an adeno-associated virus solution.
Preferably, the antibody-coupled microbeads are present in excess of the primary antibodies. The antibody-conjugated microbeads were used in excess of the primary antibodies to immobilize all primary antibodies that bind to AAV on the microbeads to prevent loss of AAV.
The present invention screens and prepares antibodies with high specificity and affinity for AAV, and the antibodies have a specific pH activity range, making them easy to use for isolation of AAV. The AAV purification kit prepared by utilizing the characteristics of the AAV antibody can rapidly purify adeno-associated virus by means of centrifugal separation (using agarose microbeads) or magnetic separation (using magnetic beads), and improves the infection titer of AAV obtained by separation.
Drawings
FIG. 1 is a statistical chart of the specific binding capacity of the nanobody AT1-3 to AAV1-8 detected by ELISA.
FIG. 2 is a graph showing the change in the binding capacity of nanobody AT2 to AAV with environmental pH.
FIG. 3 is a graph showing the binding capacity of a secondary antibody to mouse Fc as a function of environmental pH.
FIG. 4 is a statistical plot of the relative protein content in the disrupted supernatant, the isolated supernatant, and the purified AAV solution.
FIG. 5 is a comparison of gene copy number titers of AAV solutions purified from the kits of the invention and commercial kits.
FIG. 6 is a comparison of the infectious titer of AAV solutions purified from a kit of the invention and a commercially available kit.
Detailed Description
The principles and features of the present invention are described below with reference to the drawings, the examples are illustrated for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention.
1. Screening of monoclonal antibodies against AAV
The amino acid sequences of VP1, VP2 and VP3 of AAV1-13 are aligned, a conserved sequence is extracted, a synthesized sequence is cloned into microzyme for expression to obtain an AAV coat protein immunogen, and the immunogen is used for immunizing alpaca, and monoclonal antibody screening is carried out on the alpaca spleen. Finally, we screened 3 monoclonal antibodies with good specificity and affinity for all serotypes AAV1-8 (fig. 1). We have unexpectedly found in the study that the use of AT2 strain monoclonal antibodies is particularly sensitive to pH, and in a suitable buffer, the monoclonal antibodies have very high binding to AAV capsids when the pH is 7.5-9, but the antibodies have little binding to AAV capsids when the pH is reduced below 6 (fig. 2). By sequencing, the heavy chain variable region (VHH) sequence of AT2 is shown as SEQ ID NO. 1, wherein the CDR1-3 sequences are shown as SEQ ID NO. 2-4, respectively. The VHH and the mouse Fc fragment are fused and expressed to obtain a new antibody with the mouse Fc fragment and the AT2 VHH, namely the first antibody. The binding properties of the primary antibody were examined, and the results showed that they were consistent with those of the AT2 VHH, had very high specific binding capacity to AAV1-8, and were very high in the range of pH 7.5-9, but when the pH was lowered to 6 and below, the antibodies had little binding to AAV capsids.
A mouse Fc fragment is used as an immunogen to immunize alpaca, a monoclonal antibody aiming at the mouse Fc is prepared, and a second antibody AF is obtained, wherein the VHH sequence is shown as SEQ ID NO. 5, and the CDR1-3 sequences are respectively shown as SEQ ID NO. 6-8. The result of detecting the affinity of AF to the first antibody is shown in FIG. 3, and the second antibody AF has a very good affinity to the first antibody when the pH is 4-9.
Based on the properties of the two antibodies, we developed a new AAV purification kit.
2. Composition of AAV purification kit
The AAV purification kit of this example comprises the following reagents: lysis-equilibration buffer, primary antibodies, antibody-coupled microbeads, and eluate.
Wherein the antibody-coupled microbeads are magnetic beads or agarose microbeads coupled with secondary antibodies;
the lysis-balance buffer is tris buffer with pH of 8-8.5, and contains tween 20 with concentration of 0.05% -0.1% and NP40 with concentration of 0.2% -0.3%;
the eluent is citric acid buffer solution with pH of 4-5, and contains 50-100 mM sodium chloride.
3. Purification of AAV
1) Cell culture: adding DMEM medium containing 10% Fetal Bovine Serum (FBS) into culture dish, inoculating HEK293 cells, and adding 5% CO 2 Culturing at 37deg.C, and transferring every 3-5 days.
2) Viral plasmid transfection: when the cell density was 85% or more, the cells were suspended, and AAV plasmids were transferred into the cells. The plasmids transferred include: repcap plasmid (AAV-1), helper plasmid and vector plasmid, the vector plasmid carries GFP expression cassette for subsequent detection of infection efficiency of purified AAV. The transformants were cultured for 3-5 days.
3) AAV virus purification:
the cultured cell culture was centrifuged to collect the cells, resuspended in lysis-equilibration buffer, vortexed for 2-5 min to break the cells, and centrifuged to take the broken supernatant for further purification and retain a portion for subsequent functional validation experiments.
Adding a first antibody into the supernatant, and reversing and oscillating for 5-10 min to enable the first antibody to be fully combined with AAV particles; adding the antibody coupled magnetic beads into an EP tube, adding a lysis-balance buffer solution, carrying out vortex oscillation for 15s, overturning oscillation for 2 min, and removing liquid on a magnetic frame; the primary antibody-supernatant was added to the EP tube, vortexed for 15s, and then tumbled for 15-20 min to bind the AAV-primary antibody complex to the secondary antibody on the magnetic beads, anchoring the AAV on the magnetic beads. The EP tube was transferred to a magnetic rack, the liquid was removed (this step liquid was used for the subsequent functional verification experiments, for brevity, hereinafter referred to as separation supernatant), and washed twice with lysis-equilibration buffer. Then adding the eluent, shaking for 15s by vortex, and turning over and shaking for 2-5 min. Then the supernatant was aspirated onto a magnetic rack to obtain purified AAV solution. The amounts of magnetic beads and primary antibodies should be matched so that all primary antibodies can be adsorbed on the magnetic beads in order to prevent competition between the antibodies that do not bind AAV and the antibodies that bind AAV.
4. Functional verification of AAV purification kit
The AAV solution obtained by crushing the supernatant, separating the supernatant and purifying was subjected to quantitative protein detection, and the protein content of the crushed supernatant was normalized, and the result is shown in FIG. 4, wherein about 85-90% of the protein was removed by the purification. The AAV solution obtained by purification has only 3 protein bands corresponding to VP1-3 respectively in electrophoresis, which is detected by SDS-PAGE electrophoresis. Therefore, the kit can remove impurity proteins and obtain AAV solution with very high purity.
And (3) determining the titer of the AAV solution obtained by purification, carrying out gradient dilution on the AAV solution, and determining the gene copy number titer of the AAV solution by a fluorescent quantitative PCR method. We purchased two commercial AAV purification kits (commercial purification-1 and commercial purification-2), and the AAV solution obtained after purification according to the instructions was compared with the gene copy number titer of the AAV solution obtained by purification using the kit of the present invention. As shown in FIG. 5, the AAV solution purified by the kit of the invention has no obvious difference from the AAV solution purified by the commercially available AAV purification kit, the separated supernatant hardly contains gene copy number, the purification is relatively thorough, and the AAV recovery rate is relatively high.
The AAV solution obtained by purifying the kit is subjected to gradient dilution to infect HEK293 cells, and the infected cells can be marked according to fluorescent expression condition due to GFP expression frame contained in vector plasmid wrapped in AAVAnd the infection titer was calculated. As a result, as shown in FIG. 6, the AAV solution purified by the kit of the present invention had an infection titer of about 5X 10 7 TU/. Mu.l, significantly higher than the two commercial kits, the isolated supernatant had no infectious activity.
As can be seen from the above effect verification experiments, the AAV solution with high purity, which can be obtained by the purification kit of the present invention, has a gene copy number titer which is not much different from that of the commercially available kit. However, in terms of infectious titer, the AAV solutions purified from the kits of the invention have significantly higher infectious titers. We speculate that it is likely that the kit of the invention eliminates some minor amounts of the hybrid protein by isolating AAV using specific antibodies, making the AAV solution purified by the kit of the invention have much higher infectious titer at about the gene copy number titer than the commercial kit.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
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Claims (8)

1. An adeno-associated virus purification kit comprising a lysis-equilibration buffer, a first antibody, an anchor microbead, and an eluate;
the first antibody is a nano antibody and is provided with a heavy chain variable region and an Fc region, and the sequence of the heavy chain variable region is shown as SEQ ID NO. 1;
the antibody-conjugated microbeads are microbeads conjugated with a second antibody, which is an antibody against the Fc region;
the lysis-balancing buffer is tris buffer with pH of 8-8.5, and contains tween 20 with concentration of 0.05% -0.1% and NP40 with concentration of 0.2% -0.3%.
2. The adeno-associated virus purification kit of claim 1, wherein the eluent is a citric acid buffer having a pH of 4-5 and contains 50-100 mM sodium chloride.
3. The adeno-associated virus purification kit of claim 1, wherein the Fc region is a mouse Fc region.
4. The adeno-associated virus purification kit according to claim 3, wherein the second antibody is a nanobody, and the heavy chain variable region sequence is shown in SEQ ID NO. 5.
5. The adeno-associated virus purification kit of claim 1, wherein the antibody-coupled microbeads are agarose microbeads or magnetic beads.
6. A method of purifying an adeno-associated virus comprising the step of purifying an adeno-associated virus using the adeno-associated virus purification kit of any one of claims 1-5.
7. The method according to claim 6, comprising the steps of:
s1: collecting cells containing adeno-associated virus, disrupting the cells using the lysis-equilibration buffer, and centrifuging to obtain a supernatant containing adeno-associated virus;
s2: adding the first antibody into the supernatant, and uniformly mixing to obtain a first antibody-supernatant;
s3: adding the antibody-coupled microbeads into the first antibody-supernatant, and uniformly mixing;
s4: separating the antibody-conjugated microbeads from the first antibody-supernatant;
s5: eluting the antibody-coupled microbeads with the eluent to obtain an adeno-associated virus solution.
8. The method of claim 7, wherein the antibody-coupled microbeads are in excess of the first antibodies.
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